We previously showed using microarray profiling biotechnology that stepwise transformation and metastatic progression of SCC in a murine model results in the expression of gene programs related to the signal transcription factor NF-kappaB. Inhibition of NF-kappaB modulated over half the up or down-regulated genes differentially expressed, and attenuated the malignant phenotype, indicating it may be a critical target for prevention or therapy of head and neck cancer. Gene expression profiling and bioinformatic analysis of the promoters of gene clusters differentially expressed in human HNSCC provided evidence for increased prevalence of binding motifs for NF-kappaB as well as other signal transcription factors, such as p53, AP-1, STAT3 and EGR-1 (Yan et al, Genome Biology, 2007). NF-kappaB, p53, AP-1, STAT3 and EGR-1 activation has previously been associated with pathogenesis and therapeutic resistance, and the subsets expressing wt or mt 53 have been reported to differ in response to chemotherapy. These observations suggested the hypothesis that key alterations in a network of signal transcription factors can interact in determining gene expression and development of HNSCC of differing malignant potential and sensitivity or resistance to therapy. One relationship recently identified was between NF-kB member c-REL and p53 members p53, p63 and p73. This led to demonstration that cytokine TNF induced cREL interacts with p63, displacing p73 from growth arrest and apoptotic genes and the nucleus of HNSCC (Lu et al, Cancer Res, 2011). Novel p63, cREL as well as classical p53 and NF-kB sites were defined in a broader set of cancer genes and validated by ChIP assay (Yang et al, Cancer Res, 2011). ChIP sequencing of global gene expression regulated by cREL, p63 and p73 in HNSCC is underway, and has revealed the importance of these interactions genome-wide (Lu and Si et al, manuscript in preparation). We are developing use of high throughput oligonucleotide based enzyme linked assays for transcription factor binding to oligos to validate putative promoter sites identified by ChIP sequencing . Next generation RNA and DNA sequencing of HNSCC has been undertaken to identify important genetic and microRNA drivers that regulate broader changes in gene expression and malignant phenotype. We have identified a candidate family of miRNAs regulated by NF-kB that repress TP53 expression (Saleh et al, manuscript in preparation). Additionally, we have identified mutations and changes in expression of genes important in tumor subsets that are altered in established HNSCC lines that may be useful as models for studies of the molecular function of these alterations and as targets for therapy (Yang and Si et al, manuscript in preparation).